E. coli deficient in dUTPase (dut mutants) and uracil-DNA glycosylase (ung mutants) incorporate significant amounts of uracil in place of thymine into bacterial and phage DNA. The aim of this project is to understand how uracil incorporation affects the metabolism and function of DNA. There are two major subprojects in this research program. The first is to study nucleotide and DNA metabolism. The goals of this research are to understand a) the factors influencing uracil incorporation into DNA; and b) the enzymatic pathway for excising and repairing uracil-containing DNA. This work requires studying mutants defective in nucleotide metabolism. The mutations will be further characterized and the interactions of mutants will be analyzed. Such studies should allow an increase in uracil content of DNA and may improve the viability of U-DNA bacteria or otherwise yield an understanding of how U-DNA affects the viability of such bacteria. The second major subproject is to study the effects of uracil-DNA on the rest of metabolism. How do changes in U-DNA structure and interactions lead to changes in nucleic acid functions? The effects of U-DNA on gene expression will be studied by comparing U-DNA bacteria and phage to their T-DNA counterparts. Specialized transducing phage will be used because the U-DNA synthesis step is dissociated from studies of its effects on beta-galactosidase synthesis. The uracil-DNA glycosylase gene will be cloned into a multicopy plasmid to construct a strain that overproduces the enzyme activity (for enzymological studies and studies on the effect of this enzyme on the mutation rate). The U-DNA phage will be used in a phage reactivation system to evaluate the importance of DNA repair enzymes in base-excision repair and to consider the possible participation of other enzymes not recognized as repair enzymes. Work along these lines should yield insight into why RNA contains uracil while the DNA of all cells contains thymine.